The premise of this application is that cumulus cells produce paracrine regulators crucial for normal oocyte development, and that disruption of their function will lead to the production of oocytes that cannot undergo fertilization or mbryonic development. This project will use signal sequence trap (SST) methodology to identify and characterize paracrine factors produced by cumulus cells that promote oocyte development. In addition, the SST will identify transmembrane receptors expressed by cumulus cells. These receptors may be necessary for cumulus cell functions essential for oocyte development or ovulation. Specific Aim 1 is to produce and screen a cumulus cell SST library in order to isolate cumulus cell-secreted factors and transmembrane receptors. Specific Aim 2 is to characterize and clone cumulus cell cDNAs that contain putative signal peptide sequences. Novel sequences will be further characterized by Northern blot and RNase protection analyses using multiple tissues and by in situ hybridization using ovaries at key developmental stages to establish specificity of cellular expression. cDNA inserts exhibiting cumulus cell-specific expression will be used to isolate full-length cDNAs from a cumulus cell cDNA library. Sequence analysis of the full-length cDNAs will be performed, and novel sequences will be used for further studies, including the isolation of genomic clones from 129/SvEv mouse libraries. Specific Aim 3 is to assess the function of putative cumulus cell-expressed ligands and receptors, and to define their participation in the oocyte-granulosa cell regulatory loop. Null alleles of genes that encode novel cumulus cell-specific proteins (identified in Specific Aim 2) will be produced through collaboration with the Center's Transgenic Knockout Core Facility. Heterozygous mice will be bred to homozygosity to assess the effects of the null mutations on fertility. In addition, ovarian histology will be evaluated with an emphasis on follicle development, cumulus cells, and oocytes. At the molecular level, the expression of potential downstream marker genes in oocytes will be investigated. The effects of the null mutations and cumulus cell-expressed recombinant proteins on oocyte and cumulus cell function in vitro, including the ability of oocyte to resume and complete meiosis, undergo fertilization, and complete preimplantation development, will be determined. Success in these studies will generate exciting new knowledge of the fundamental mechanisms that govern the development of both oocytes and the somatic components of follicles, as well as provide a plethora of new targets for fertility control.